巴戟天ISSR体系的建立与优化  

Establishment and Optimization of ISSR-PCR Reaction System of Morinda officinalis How

作　　者：刘颖嘉[1] 黄宇[1] 荣俊冬[1] 何天友[1] 胡迪科[1] 郑郁善[1] 

机构地区：[1]福建农林大学工业原料林研究所,福州350002

出　　处：《种子》2011年第9期1-4,共4页Seed

基　　金：国家科技支撑计划(2009BAI73B01);福建省科技重大专项(2004YZ02-05);福建省中药材GAP工程技术研究中心(2008Y2001)

摘　　要：以巴戟天叶片提取的基因组DNA为材料,对影响ISSR-PCR扩增效果的一些因素,如dNTPs浓度、Mg2+浓度、TaqDNA聚合酶用量、引物用量、模板DNA用量以及退火温度等指标进行筛选和优化,确立了可用于巴戟天的ISSR-PCR分析最适宜的PCR反应条件,即20μl PCR反应体积中含0.2 mmol/L dNTPs2,.0 mmol/L Mg2+1,.0 U Taq DNA聚合酶,0.5μmol/L引物5,0 ng模板DNA。PCR扩增程序:94℃预变性5 min9,4℃变性30 s,53.4℃退火45 s,72℃延伸1 min,45个循环7,2℃延伸10 min4,℃保存。应用该ISSR体系对6份巴戟天种质进行了扩增,证实了该体系的适用性和稳定性。Taking genomic DNA extracted from Morinda officinalis How. leaves as the experimental material, the factors which affected the ISSR-PCR amplification such as suitable concentration for dNTPs and Mg2＋ , the dosage for Taq DNA polymerase, the primer, template DNA and annealing temperature were selected and optimized. The results showed that the suitable PCR conditions for ISSR-PCR of Morinda officinalis How. were as follows ： In the 20 H1 PCR reaction volume, 0.2 mmol/L dNTPs, 2. 0 mmol/L Mg2＋ , 1. 0 U TaqDNA polymerase ,0.5 mol/L primer,50 ng template DNA. The optimal PCR amplification process was：5 minutes at 94 ℃ for predenaturation, then followed by 45 cycles, each with 30 seconds at 94 ℃ for denaturation, 45 seconds at 53.4℃ for annealing, 1 minute at 72℃ for extension, finally extension at 72℃ for 10 minutes and holding the samples at 4℃. The system was applied in the amplification of six varieties of Morinda officinalis How. ,indicated the suitability and stability of the system.

关 键 词：巴戟天 ISSR—PCR 体系优化 

分 类 号：S567.19[农业科学—中草药栽培]